52 and 19 years, respectively [18]. It required a pandemic of this scale to force these

new technologies into being combined with unprecedented international cooperation

and funding. In fact, the phase IV trials for encapsulated mRNA vaccines represent

the biggest trials ever for a nanomedicine.

12.4.1.1

mRNA Vaccine Design

To understand the potential these technologies hold, it is important to discuss their

design in greater detail. Due to the instability of RNA molecules, they cannot be

injected naked into the human body. Therefore, they are encapsulated in a vector

such as a lipid nanoparticle (LNP). Whereas, DNA, due to its increased stability,

can be injected as a free plasmid. The LNP not only serves to protect the mRNA

from premature degradation, but it also facilitates its entry into the target cells. If the

mRNA were not encapsulated, it would be rapidly degraded by the nucleases in the

body [29]. The goal is to use nanocarriers that are non-toxic and non-immunogenic

which would allow for repeated dosing [25].

The mRNA molecule itself also needs to be specially designed to maximize the

amount and quality of antigen produced. There should be a 5′ cap, 5′ UTR, ORFs, 3′

UTR, and a poly-A tail. These sequences are specially designed to prevent reverse

binding of the RNA molecule and to increase stability of the molecule [7]. Lastly,

the ORFs are optimized to amplify translation of the antigen.

Once the mRNA reaches the cell, it can be directly translated in the cytoplasm by

host ribosomes. This contrasts with DNA plasmids, which must be translocated to

the nucleus prior to transcription, a more complicated process that may impair

protein expression. However, the half-life of DNA expression in the nucleus is

significantly longer than mRNA in the cytoplasm [18]. This may result in a longer-

lived immunity from DNA vaccines, although DNA vaccines have been shown to

have generally poor, mostly cell-mediated, immunity [26].

Another major advantage of nucleic acid vaccines is that once the protein is

translated, any post-translational modifications that normally occur in a natural in-

fection can take place. This serves to further increase the specificity of the immune

response. For example, the S protein has 22 glycosylation sites [30]. Following these

post-translational modifications, the S protein is transported to the cell membrane

where it is presented as a membrane-bound antigen at the cell surface. It will then be

recognized by T-cells and B-cells via MHC presentation [28].

In summary, following injection and local inflammation, the LNP-encapsulated

mRNA is taken up by antigen-presenting cells, which then migrate to the draining

lymph nodes. Upon translation and presentation of the antigen, toll-like receptors

are activated leading to cytokine production. This eventually leads to a robust T-cell

response with Th1 type CD4+ cells and CD8+ cells. The CD4+ T-cells then activate

the antigen-specific B-cells, leading to their differentiation into plasma cells and

antibody production [31].

There are currently two mRNA vaccines that are approved for use, namely Pfizer/

BioNTech (PB) and Moderna. The Moderna vaccine is also known as mRNA-1273,

because it codes for the entire 1273 amino acid sequence of the S protein. PB, on the

other hand, developed two different vaccines BNT163b1 and BNT162b2, which code

for the RBD of S1 and the full-length S-protein in the prefusion conformation,

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Bioprocessing of Viral Vaccines